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Abstract Loss of function mutations in the gene encoding dystrophin elicits a hypersensitive fear response in mice and humans. In the dystrophin-deficient mdx mouse, this behaviour is partially protected by oestrogen, but the mechanistic basis for this protection is unknown. Here, we show that female mdx mice remain normotensive during restraint stress compared to a hypotensive and hypertensive response in male mdx and male/female wildtype mice, respectively. Partial dystrophin expression in female mdx mice (heterozygous) also elicited a hypertensive response. Ovariectomized (OVX) female mdx mice were used to explain the normotensive response to stress. OVX lowered skeletal muscle mass and lowered the adrenal mass and zona glomerulosa area (aldosterone synthesis) in female mdx mice. During a restraint stress, OVX dampened aldosterone synthesis and lowered the corticosterone:11-dehydrocorticosterone. All OVX-induced changes were restored with replacement of oestradiol, except that oestradiol lowered the zona fasciculata area of the adrenal gland, dampened corticosterone synthesis but increased cortisol synthesis. These data suggest that oestrogen partially attenuates the unconditioned fear response in mdx mice via adrenal and vascular function. It also suggests that partial dystrophin restoration in a dystrophin-deficient vertebrate is an effective approach to develop an appropriate hypertensive response to stress.

Involuntary celibates (incels) are part of the online ‘manosphere’ and have been widely discussed in contemporary media in recent years due to their involvement in several offline mass murders. This article presents empirical data that specifically map aspects of the incel worldview: the ‘black pill’. Analysis of online discussion forums demonstrates how incels believe society is ordered through a hetero-patriarchal racial hierarchy and justify their sexlessness through beliefs rooted in biological determinism and victimisation by women and feminism. It is argued that the black pill is a disciplinary device that aids in building a digital counter-public that engenders a collective incel identity. Further, the article argues that the black pill produces a form of ‘stochastic terrorism’ in which users interpret its spectrum of beliefs to enact harms from online gender-based hate speech through terrorist violence in the offline world. As a point of departure, the article argues that incel counter-publics transcend the false distinctions between online and offline; both ‘worlds’ contribute to the (re)production of incel anti-feminism and misogyny.

Abstract Background Dystrophin deficiency sensitizes skeletal muscle of mice to eccentric contraction (ECC)-induced strength loss. ECC protocols distinguish dystrophin-deficient from healthy, wild type muscle, and test the efficacy of therapeutics for Duchenne muscular dystrophy (DMD). However, given the large lab-to-lab variability in ECC-induced strength loss of dystrophin-deficient mouse skeletal muscle (10–95%), mechanical factors of the contraction likely impact the degree of loss. Therefore, the purpose of this study was to evaluate the extent to which mechanical variables impact sensitivity of dystrophin-deficient mouse skeletal muscle to ECC. Methods We completed ex vivo and in vivo muscle preparations of the dystrophin-deficient mdx mouse and designed ECC protocols within physiological ranges of contractile parameters (length change, velocity, contraction duration, and stimulation frequencies). To determine whether these contractile parameters affected known factors associated with ECC-induced strength loss, we measured sarcolemmal damage after ECC as well as strength loss in the presence of the antioxidant N-acetylcysteine (NAC) and small molecule calcium modulators that increase SERCA activity (DS-11966966 and CDN1163) or lower calcium leak from the ryanodine receptor (Chloroxine and Myricetin). Results The magnitude of length change, work, and stimulation duration ex vivo and in vivo of an ECC were the most important determinants of strength loss in mdx muscle. Passive lengthening and submaximal stimulations did not induce strength loss. We further showed that sarcolemmal permeability was associated with muscle length change, but it only accounted for a minimal fraction (21%) of the total strength loss (70%). The magnitude of length change also significantly influenced the degree to which NAC and small molecule calcium modulators protected against ECC-induced strength loss. Conclusions These results indicate that ECC-induced strength loss of mdx skeletal muscle is dependent on the mechanical properties of the contraction and that mdx muscle is insensitive to ECC at submaximal stimulation frequencies. Rigorous design of ECC protocols is critical for effective use of strength loss as a readout in evaluating potential therapeutics for muscular dystrophy.

Skeletal muscle dysfunction may contribute to the progression and severity of amyotrophic lateral sclerosis (ALS). In the present study, we characterized the skeletal muscle pathophysiology in an inducible transgenic mouse model (rNLS8) that develops a TAR-DNA binding protein (TDP-43) proteinopathy and ALS-like neuropathology and disease progression; representative of >90% of all familial and sporadic ALS cases. As we previously observed elevated levels of miR-23a in skeletal muscle of patients with familial and sporadic ALS, we also investigated the effect of miR-23a suppression on skeletal muscle pathophysiology and disease severity in rNLS8 mice. Five weeks after disease onset TDP-43 protein accumulation was observed in tibialis anterior (TA), quadriceps (QUAD) and diaphragm muscle lysates and associated with skeletal muscle atrophy. In the TA muscle TDP-43 was detected in muscle fibres that appeared atrophied and angular in appearance and that also contained β-amyloid aggregates. These fibres were also positive for neural cell adhesion molecule (NCAM), but not embryonic myosin heavy chain (eMHC), indicating TDP-43/ β-amyloid localization in denervated muscle fibres. There was an upregulation of genes associated with myogenesis and NMJ degeneration and a decrease in the MURF1 atrophy-related protein in skeletal muscle. Suppression of miR-23a impaired rotarod performance and grip strength and accelerated body weight loss during early stages of disease progression. This was associated with increased AchRα mRNA expression and decreased protein levels of PGC-1α. The TDP-43 proteinopathy-induced impairment of whole body and skeletal muscle functional performance is associated with muscle wasting and elevated myogenic and NMJ stress markers. Suppressing miR-23a in the rNLS8 mouse model of ALS contributes to an early acceleration of disease progression as measured by decline in motor function.

Weakness and atrophy are key features of Duchenne muscular dystrophy (DMD). Dystrophin is one of the many proteins within the dystrophin glycoprotein complex (DGC) that maintains plasmalemmal integrity and cellular homeostasis. The dystrophin-deficient mdx mouse is also predisposed to weakness, particularly when subjected to eccentric (ECC) contractions due to electrophysiological dysfunction of the plasmalemma. Here, we determined if maintenance of plasmalemmal excitability during and after a bout of ECC contractions is dependent on intact and functional DGCs rather than, solely, dystrophin expression. Wild-type (WT) and dystrophic mice (mdx, mL172H and Sgcb−/− mimicking Duchenne, Becker and Limb-girdle Type 2E muscular dystrophies, respectively) with varying levels of dystrophin and DGC functionality performed 50 maximal ECC contractions with simultaneous torque and electromyographic measurements (M-wave root-mean-square, M-wave RMS). ECC contractions caused all mouse lines to lose torque (p

Objective: Phosphatidylethanolamine (PtdEtn) is a major phospholipid in mammals. It is synthesized via two pathways, the CDP-ethanolamine pathway in the endoplasmic reticulum and the phosphatidylserine (PtdSer) decarboxylase (PSD) pathway in the mitochondria. While the CDP-ethanolamine pathway is considered the major route for PtdEtn synthesis in most mammalian tissues, little is known about the importance of the PSD pathway in vivo, especially in tissues enriched with mitochondria such as skeletal muscle. Therefore, we aimed to examine the role of the mitochondrial PSD pathway in regulating PtdEtn homeostasis in skeletal muscle in vivo. Methods: To determine the functional significance of this pathway in skeletal muscle in vivo, an adeno-associated viral vector approach was employed to knockdown PSD expression in skeletal muscle of adult mice. Muscle lipid and metabolite profiling was performed using mass spectrometry. Results: PSD knockdown disrupted muscle phospholipid homeostasis leading to an ∼25% reduction in PtdEtn and an ∼45% increase in PtdSer content. This was accompanied by the development of a severe myopathy, evident by a 40% loss in muscle mass as well as extensive myofiber damage as shown by increased DNA synthesis and central nucleation. In addition, PSD knockdown caused marked accumulation of abnormally appearing mitochondria that exhibited severely disrupted inner membrane integrity and reduced OXPHOS protein content. Conclusions: The PSD pathway has a significant role in maintaining phospholipid homeostasis in adult skeletal muscle. Moreover, PSD is essential for maintenance of mitochondrial integrity and skeletal muscle mass. Keywords: Phospholipids, Phosphatidylethanolamine, Phosphatidylserine, Mitochondria, Skeletal muscle, Atrophy

The highly ordered cortical microtubule lattice of skeletal muscle is disorganized in dystrophin-deficient mdx mice. Implicated mechanisms include loss of dystrophin binding, altered α-tubulin posttranslational modification, expression of a β-tubulin involved in regeneration, and reactive oxygen species (ROS). Here we show that the transverse microtubules in mdx muscle expressing miniaturized dystrophins are rapidly lost after eccentric contraction. Analysis of mdx lines expressing different dystrophin constructs demonstrate that spectrin-like repeats R4-15 and R20-23 were required for mechanically stable microtubules. Microtubule loss was prevented by the non-specific antioxidant N-acetylcysteine while inhibition of NADPH oxidase 2 had only a partial effect, suggesting that ROS from multiple sources mediate the rapid loss of transverse microtubules after eccentric contraction. Finally, ablation of α-dystrobrevin, β- or γ-cytoplasmic actin phenocopied the transverse microtubule instability of miniaturized dystrophins. Our data demonstrate that multiple dystrophin domains, α-dystrobrevin and cytoplasmic actins are necessary for mechanically stable microtubules.

Background: Duchenne muscular dystrophy (DMD) is caused by the loss of dystrophin. Severe and ultimately lethal, DMD progresses relatively slowly in that patients become wheelchair bound only around age twelve with a survival expectancy reaching the third decade of life. Methods: The mildly-affected mdx mouse model of DMD, and transgenic DysΔMTB-mdx and Fiona-mdx mice expressing dystrophin or utrophin, respectively, were exposed to either mild (scruffing) or severe (subordination stress) stress paradigms and profiled for their behavioral and physiological responses. A subgroup of mdx mice exposed to subordination stress were pretreated with the beta-blocker metoprolol. Findings: Subordination stress caused lethality in ∼30% of mdx mice within 24 h and ∼70% lethality within 48 h, which was not rescued by metoprolol. Lethality was associated with heart damage, waddling gait and hypo-locomotion, as well as marked up-regulation of the hypothalamus-pituitary-adrenocortical axis. A novel cardiovascular phenotype emerged in mdx mice, in that scruffing caused a transient drop in arterial pressure, while subordination stress caused severe and sustained hypotension with concurrent tachycardia. Transgenic expression of dystrophin or utrophin in skeletal muscle protected mdx mice from scruffing and social stress-induced responses including mortality. Interpretation: We have identified a robust new stress phenotype in the otherwise mildly affected mdx mouse that suggests relatively benign handling may impact the outcome of behavioural experiments, but which should also expedite the knowledge-based therapy development for DMD. Funding: Greg Marzolf Jr. Foundation, Summer's Wish Fund, NIAMS, Muscular Dystrophy Association, University of Minnesota and John and Cheri Gunvalson Trust.

In the mdx mouse model of Duchenne muscular dystrophy, muscle contractions lead to force loss, which is attributed to myofibre damage. Here, the authors show that force loss is instead mediated by a redox circuit involving NOX2, PROX1, myoglobin and cytoplasmic actins, and suggest that it may be a protective mechanism to prevent excessive contraction-induced myofibre damage.

Neopterin has been extensively used as a clinical marker of immune activation during inflammation in a wide range of conditions and stresses. However, the analysis of neopterin alone neglects the cellular reactions that generate it in response to interferon-γ. Neopterin is the oxidation product of 7,8-dihydroneopterin, which is a potent antioxidant generated by interferon-γ-activated macrophages. 7,8-Dihydroneopterin can protect macrophage cells from a range of oxidants through a scavenging reaction that generates either neopterin or dihydroxanthopterin, depending on the oxidant. Therefore, plasma and urinary neopterin levels are dependent on both macrophage activation to generate 7,8-dihydroneopterin and subsequent oxidation to neopterin. This relationship is clearly shown in studies of exercise and impact-induced injury during intense contact sport. Here, we argue that neopterin and total neopterin, which is the combined value of 7,8-dihydroneopterin and neopterin, could provide a more comprehensive analysis of clinical inflammation than neopterin alone.

Simple Summary: Breast cancer patients often receive anti-hormonal treatment if their tumor is positive for the Estrogen Receptor (ER), but tumors may become resistant to this therapy and still metastasize. We studied 101 of such metastatic lesions and investigated these lesions for mutated genes and mutation patterns, in combination with the level of expression of relevant genes. Our aim was to better understand the mechanisms that are involved in the resistance to anti-hormonal treatment. The analyses showed two distinct groups of patients, each with specific mutations. One group clearly showed an ongoing, active ER and its associated signal route; these patients probably still would benefit from ER-targeting agents. We advocate for combining mutation and expression analyses on metastatic lesions, to maximize the group of patients that still may benefit from existing or new anti-hormonal treatments targeting ER or its signaling network. Mutations in the estrogen receptor gene (ESR1), its transcriptional regulators, and the mitogen-activated protein kinase (MAPK) pathway are enriched in patients with endocrine-resistant metastatic breast cancer (MBC). Here, we integrated whole genome sequencing with RNA sequencing data from the same samples of 101 ER-positive/HER2-negative MBC patients who underwent a tumor biopsy prior to the start of a new line of treatment for MBC (CPCT-02 study, NCT01855477) to analyze the downstream effects of DNA alterations previously linked to endocrine resistance, thereby gaining a better understanding of the associated mechanisms. Hierarchical clustering was performed using expression of ESR1 target genes. Genomic alterations at the DNA level, gene expression levels, and last administered therapy were compared between the identified clusters. Hierarchical clustering revealed two distinct clusters, one of which was characterized by increased expression of ESR1 and its target genes. Samples in this cluster were significantly enriched for mutations in ESR1 and amplifications in FGFR1 and TSPYL. Patients in the other cluster showed relatively lower expression levels of ESR1 and its target genes, comparable to ER-negative samples, and more often received endocrine therapy as their last treatment before biopsy. Genes in the MAPK-pathway, including NF1, and ESR1 transcriptional regulators were evenly distributed. In conclusion, RNA sequencing identified a subgroup of patients with clear expression of ESR1 and its downstream targets, probably still benefiting from ER-targeting agents. The lower ER expression in the other subgroup might be partially explained by ER activity still being blocked by recently administered endocrine treatment, indicating that biopsy timing relative to endocrine treatment needs to be considered when interpreting transcriptomic data. [ABSTRACT FROM AUTHOR]

We examined whether calcineurin-NFAT (nuclear factors of activated T cells) signaling plays a role in specifically directing the expression of utrophin in the synaptic compartment of muscle fibers. Immunofluorescence experiments revealed the accumulation of components of the calcineurin-NFAT signaling cascade within the postsynaptic membrane domain of the neuromuscular junction. RT-PCR analysis using synaptic vs. extrasynaptic regions of muscle fibers confirmed these findings by showing an accumulation of calcineurin transcripts within the synaptic compartment. We also examined the effect of calcineurin on utrophin gene expression. Pharmacological inhibition of calcineurin in mice with either cyclosporin A or FK506 resulted in a marked decrease in utrophin A expression at synaptic sites, whereas constitutive activation of calcineurin had the opposite effect. Mutation of the previously identified NFAT binding site in the utrophin A promoter region, followed by direct gene transfer studies in mouse muscle, led to an inhibition in the synaptic expression of a lacZ reporter gene construct. Transfection assays performed with cultured myogenic cells indicated that calcineurin acted additively with GA binding protein (GABP) to transactivate utrophin A gene expression. Because both GABP- and calcineurin-mediated pathways are targeted by peroxisome proliferator-activated receptor-[gamma]-coactivator-1[alpha] (PGC-1[alpha]), we examined whether this coactivator contributes to utrophin gene expression. In vitro and in vivo transfection experiments showed that PGC-1[alpha] alone induces transcription from the utrophin A promoter. Interestingly, this induction is largely potentiated by coexpression of PGC-1[alpha] with GABP. Together, these studies indicate that the synaptic expression of utrophin is also driven by calcineurin-NFAT signaling and occurs in conjunction with signaling events that involve GABP and PGC-1[alpha]. synaptic gene expression; Duchenne muscular dystrophy; nuclear respiratory factor 2

Quantification of tumor‐specific variants (TSVs) in cell‐free DNA is rapidly evolving as a prognostic and predictive tool in patients with cancer. Currently, both variant allele frequency (VAF) and number of mutant molecules per mL plasma are used as units of measurement to report those TSVs. However, it is unknown to what extent both units of measurement agree and what are the factors underlying an existing disagreement. To study the agreement between VAF and mutant molecules in current clinical studies, we analyzed 1116 TSVs from 338 patients identified with next‐generation sequencing (NGS) or digital droplet PCR (ddPCR). On different study cohorts, a Deming regression analysis was performed and its 95% prediction interval was used as surrogate for the limits of agreement between VAF and number of mutant molecules per mL and to identify outliers. VAF and number of mutant molecules per mL plasma yielded greater agreement when using ddPCR than NGS. In case of discordance between VAF and number of mutant molecules per mL, insufficient molecular coverage in NGS and high cell‐free DNA concentration were the main responsible factors. We propose several optimization steps needed to bring monitoring of TSVs in cell‐free DNA to its full potential. [ABSTRACT FROM AUTHOR]

In metastatic colorectal cancer, RAS and BRAF mutations cause resistance to anti‐EGFR therapies, such as cetuximab. Heterogeneity in RAS and BRAF mutations might explain nonresponse in a subset of patients receiving cetuximab. Analyzing mutations in plasma‐derived circulating tumor DNA (ctDNA) could provide a more comprehensive overview of the mutational landscape as compared to analyses of primary and/or metastatic tumor tissue. Therefore, this prospective multicenter study followed 34 patients with metastatic colorectal cancer who were tissue‐tested as RAS wild‐type (exons 2–4) during routine work‐up and received third‐line cetuximab monotherapy. BRAF mutation status was also tested but did not exclude patients from therapy. At baseline and upon disease progression, cell‐free DNA (cfDNA) was isolated for targeted next‐generation sequencing (NGS). At 8 weeks, we determined that patients had benefited from treatment. NGS of cfDNA identified three patients with RAS mutations not detected in tumor tissue during routine work‐up. Another six patients had a BRAF or rare RAS mutation in ctDNA and/or tumor tissue. Relative to patients without mutations in RAS/BRAF, patients with mutations at baseline had shorter progression‐free survival [1.8 versus 4.9 months (P < 0.001)] and overall survival [3.1 versus 9.4 months (P = 0.001)]. In patients with clinical benefit (progressive disease after 8 weeks), ctDNA testing revealed previously undetected mutations in RAS/BRAF (71%) and EGFR (47%), which often emerged polyclonally. Our results indicate that baseline NGS of ctDNA can identify additional RAS mutation carriers, which could improve patient selection for anti‐EGFR therapies. Acquired resistance, in patients with initial treatment benefit, is mainly explained by polyclonal emergence of RAS,BRAF, and EGFR mutations in ctDNA. [ABSTRACT FROM AUTHOR]

Purpose: The main objective of this preliminary analysis of the IMaging PAtients for Cancer drug selecTion (IMPACT)-renal cell cancer (RCC) study is to evaluate the lesion detection of baseline contrast-enhanced CT, [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/CT in detecting ccRCC lesions in patients with a good or intermediate prognosis metastatic clear cell renal cell carcinoma (mccRCC) according to the International Metastatic Database Consortium (IMDC) risk model. Methods: Between February 2015 and March 2018, 42 newly diagnosed mccRCC patients with good or intermediate prognosis, eligible for watchful waiting, were included. Patients underwent CT, [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/CT at baseline. Scans were independently reviewed and lesions of ≥10 mm and lymph nodes of ≥15 mm at CT were analyzed. For lesions with [89Zr]Zr-DFO-girentuximab or [18F]FDG-uptake visually exceeding background uptake, maximum standardized uptake values (SUVmax) were measured. Results: A total of 449 lesions were detected by ≥1 modality (median per patient: 7; ICR 4.25–12.75) of which 42% were in lung, 22% in lymph nodes and 10% in bone. Combined [89Zr]Zr-DFO-girentuximab-PET/CT and CT detected more lesions than CT alone: 91% (95%CI: 87–94) versus 56% (95%CI: 50–62, p = 0.001), respectively, and more than CT and [18F]FDG-PET/CT combined (84% (95%CI:79–88, p < 0.005). Both PET/CTs detected more bone and soft tissue lesions compared to CT alone. Conclusions: The addition of [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/CT to CT increases lesion detection compared to CT alone in newly diagnosed good and intermediate prognosis mccRCC patients eligible for watchful waiting. [ABSTRACT FROM AUTHOR]

The localized transcription of several muscle genes at the motor endplate is controlled by the Ets transcription factor GABP. To evaluate directly its contribution to the formation of the neuromuscular junction, we generated transgenic mice expressing a general Ets dominant-negative mutant specifically in skeletal muscle. Quantitative RT-PCR analysis demonstrated that the expression of genes containing an Ets-binding site was severely affected in the mutant mice. Conversely, the expression of other synaptic genes, including MuSK and Rapsyn, was unchanged. In these animals, muscles expressing the mutant transcription factor developed normally, but examination of the post-synaptic morphology revealed marked alterations of both the primary gutters and secondary folds of the neuro-muscular junction. Our results demonstrate that Ets transcription factors are crucial for the normal formation of the neuro-muscular junction. They further show that Ets-independent mechanisms control the synaptic expression of a distinct set of synaptic genes. [ABSTRACT FROM AUTHOR]

Presents information on a study which examined whether local transcriptional activation of the acetylcholinesterase (AChE) gene contributes to the synaptic accumulation of AChE transcripts. Methods of the study; Results and discussion.

Examines the role of nerve-derived trophic factors and DNA elements in controlling expression of genes encoding synaptic proteins in myoneural junction. Significance of acetylcholinesterase (AChE) in neuromuscular junction; Characteristics of skeletal muscle fibers; Factors influencing the regulation of transcriptional activation of the AChE gene.

Patients with BRAF mutated (BRAF-mt) metastatic melanoma benefit significantly from treatment with BRAF inhibitors. Currently, the BRAF status is determined on archival tumor tissue or on fresh tumor tissue from an invasive biopsy. The aim of this study was to evaluate whether radiomics can predict the BRAF status in a non-invasive manner. Patients with melanoma lung metastases, known BRAF status, and a pretreatment computed tomography scan were included. After semi-automatic annotation of the lung lesions (maximum two per patient), 540 radiomics features were extracted. A chest radiologist scored all segmented lung lesions according to the Lung Image Database Consortium (LIDC) criteria. Univariate analysis was performed to assess the predictive value of each feature for BRAF mutation status. A combination of various machine learning methods was used to develop BRAF decision models based on the radiomics features and LIDC criteria. A total of 169 lung lesions from 103 patients (51 BRAF-mt; 52 BRAF wild type) were included. There were no features with a significant discriminative value in the univariate analysis. Models based on radiomics features and LIDC criteria both performed as poorly as guessing. Hence, the BRAF mutation status in melanoma lung metastases cannot be predicted using radiomics features or visually scored LIDC criteria. [ABSTRACT FROM AUTHOR]